Ice recrystallisation inhibition protein or antifreeze proteins from deschampsia, and festuca species of grass

ABSTRACT

The present invention relates to nucleic acids or nucleic acid fragments encoding amino acid sequences for polypeptides involved in tolerance to freezing and/or low temperature stress in plants. More particularly, the present invention relates to nucleic acids or nucleic acid fragments encoding amino acid sequences for ice recrystallisation inhibition proteins (IRIPs) in plants, and the use thereof for the modification of plant response to freezing and/or low temperature stress. Even more particularly, the present invention relates to polypeptides involved in tolerance to freezing and/or low temperature stress in  Deschampsia  and  Festuca  species.

The present invention relates to nucleic acids or nucleic acid fragmentsencoding amino acid sequences for polypeptides involved in tolerance tofreezing and/or low temperature stress in plants. More particularly, thepresent invention relates to nucleic acids or nucleic acid fragmentsencoding amino acid sequences for ice recrystallisation inhibitionproteins (IRIPs) in plants, and the use thereof for the modification ofplant response to freezing and/or low temperature stress. Even moreparticularly, the present invention relates to polypeptides involved intolerance to freezing and/or low temperature stress in Deschampsia andLolium species.

Plants have evolved a range of physiological and biochemical responsesto freezing and low temperature stress. As a consequence ofpoikilothermy many plant species are tolerant of temperature extremes,including exposure to sub-zero temperatures. Sub-zero temperaturesnegatively impact on plant cells in many ways. As temperatures dropbelow freezing ice crystal formation initially takes placeextracellularly, in the apoplasm. This leads to an elevation ofintracellular solute concentration as water is lost by osmosis to theextracellular ice, resulting in severe dehydration. Desiccation, wherebyas much as 90% of intracellular water can be lost at −10° C., inducesmultiple forms of membrane damage. Furthermore, extracellular iceobstructs gas and solute exchange, and growing ice crystals causeplasmolysis.

Plants and other organisms that are exposed to subzero temperatures haveevolved varied mechanisms to confer tolerance to freezing stressincluding deployment of variant isozymes, synthesis of osmoprotectantsand compatible solutes, and modification of membrane lipid composition.A particular characteristic of tolerance to freezing, and to temperaturestresses in general, is the phenomenon of acquired tolerance. Forfreezing stress this is termed cold acclimation, whereby a transition tolow, non-freezing temperature can confer tolerance of subsequentexposure to otherwise lethal subzero temperatures.

A common response of plant and other species with tolerance to subzerotemperatures and freezing is the expression of anti-freeze proteins(AFPs). AFPs have an affinity for ice, by virtue of structuralcomplementarity, thereby inhibiting its growth. Adsorbtion of AFPs ontoice surfaces has two distinct effects: thermal hysteresis (TH) andrecrystallisation inhibition (RI). TH results from a noncolligativefreezing point depression as ice front growth becomes restricted tosterically unfavourable spaces between AFPs. This broadens the gapbetween the melting and freezing points of ice, and this range is themeasure of TH. AFPs mediate the effect of RI by interfering with themigration of ice boundaries which normally thermodynamically favour thecreation of large, ice crystals at the expense of smaller ones. Thus RIactivity limits the growth of large ice crystals that have the potentialto puncture cell walls and membranes and cause plasmolysis. RI activityhas been identified in extracts from a limited number of plant species,and the nucleotide sequence of one ice recrystallisation inhibitionprotein (IRIP) conferring such activity has been reported from Loliumperenne.

Antarctic hair grass Deschampsia antarctica is one of only twoangiosperms to have overcome the geographical and environmentalimpediments to colonising the Antarctic continent. It grows infavourable locations along the western coast of the Antarctic Peninsula.D. antarctica is an over-wintering species with a short growing seasonthat at Palmer Station (64°47′S), is typically November to March. Inrespect of low temperature stress, on Léonie Island in northernMarguerite Bay (67°36′S) towards the southern limit of distribution ofD. antarctica, air temperatures below −30° C. have been recorded duringthe austral winter. During the growing season, when plants are mostvulnerable to freezing stress, episodes of temperatures down to −15° C.can occur early in the growing season. D. antarctica has a welldeveloped cold-acclimation response, and significant cellular damageonly occurs in plants exposed to temperatures substantially below thoseat which they freeze.

Despite D. antarctica's well developed freezing tolerance no biochemicalor physiological mechanisms have been identified that can coherentlyaccount for this capacity.

There is a need for materials useful in modifying the tolerance tofreezing and low temperature stress in a wide range of plants, and formethods for their use.

It is an object of the present invention to overcome, or at leastalleviate, one or more of the difficulties or deficiencies associatedwith the prior art.

In one aspect, the present invention provides substantially purified orisolated nucleic acids or nucleic acid fragments encoding IRIPs from aDeschampsia species, preferably Antarctic hair-grass, Deschampsiaantarctica, or functionally active fragments or variants thereof.

In a preferred embodiment of this aspect of the invention, thesubstantially purified or isolated nucleic acid or nucleic acid fragmentincludes a nucleotide sequence selected from the group consisting of (a)sequences shown in FIGS. 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 and 24hereto; (b) complements of the sequences recited in (a); (c) sequencesantisense to the sequences recited in (a) and (b); (d) functionallyactive fragments and variants of the sequences recited in (a), (b) and(c); and (e) RNA sequences corresponding to the sequences recited in(a), (b), (c) and (d).

In another aspect, the present invention provides substantially purifiedor isolated nucleic acids or nucleic acid fragments encoding IRIPs froma ryegrass (Lolium) or fescue (Festuca) species. These species may be ofany suitable type, including Italian or annual ryegrass, perennialryegrass, tall fescue, meadow fescue and red fescue. Preferably thespecies is a ryegrass, more preferably perennial ryegrass (L. perenne).

In a preferred embodiment of this aspect of the invention, thesubstantially purified or isolated nucleic acid or nucleic acid fragmentincludes a nucleotide sequence selected from the group consisting of (a)sequences shown in FIGS. 26, 27, 29 and 30 hereto; (b) complements ofthe sequences recited in (a); (c) sequences antisense to the sequencesrecited in (a) and (b); (d) functionally active fragments and variantsof the sequences recited in (a), (b) and (c); and (e) RNA sequencescorresponding to the sequences recited in (a), (b), (c) and (d).

The present invention provides substantially purified or isolatednucleic acids or nucleic acid fragments encoding amino acid sequencesfor a class of polypeptides which are related to IRIP or functionallyactive fragments or variants thereof. Such proteins are referred toherein as IRIP-like. The genes which encode these polypeptides areexpressed in a similar manner to IRIP. The invention also encompassesfunctionally active fragments and variants of nucleic acids encodingsuch polypeptides.

The individual or simultaneous enhancement or otherwise manipulation ofIRIP or like gene activities in plants may enhance or otherwise alterthe freezing and/or low temperature tolerance of plants.

The modification of plant freezing and/or low temperature tolerancebased on the individual or simultaneous enhancement or otherwisemanipulation of IRIP or like gene activities in plants has significantconsequences for a range of applications in plant production and plantprotection. For example, it has applications in increasing the range andproductivity of plants.

Methods for the modification of plant freezing and/or low temperaturetolerance may facilitate the production of, for example, plants withenhanced tolerance of freezing and/or low temperature stress.

Nucleic acids according to the invention may be full-length genes orpart thereof, and are also referred to as “nucleic acid fragments” and“nucleotide sequences” in this specification.

The nucleic acid or nucleic acid fragment may be of any suitable typeand includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA)that is single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases, and combinations thereof.

Such nucleic acid fragments could be assembled to form a consensuscontig.

In a further aspect of the present invention there is provided asubstantially purified or isolated regulatory element from a Deschampsiaspecies, preferably Deschampsia antarctica, said regulatory elementbeing capable of modifying plant response to freezing and/or lowtemperature stress.

More preferably the regulatory element includes a nucleotide sequenceselected from the groups consisting of (a) sequences shown in FIGS. 32and 33 hereto; (b) complements of the sequences recited in (a); and (c)functionally active fragments and variants of the sequences recited in(a) and (b).

In a further aspect of the present invention there is provided asubstantially purified or isolated regulatory element from a Lolium orFestuca species, preferably Lolium perenne, said regulatory elementbeing capable of modifying plant response to freezing and/or lowtemperature stress.

More preferably the regulatory element includes a nucleotide sequenceselected from the group consisting of (a) sequence shown in FIG. 34hereto; (b) complement of the sequence recited in (a) and (c)functionally active fragments and variants of the sequences recited in(a) and (b).

Preferably the regulatory element is a promoter.

Preferably the regulatory element is isolated from an IRIP nucleic acidor nucleic acid fragment.

As used herein, the term IRIP-like relates to polypeptides that areproduced in the plant in substantially the same organs and atsubstantially the same developmental stages as IRIP.

The term “isolated” means that the material is removed from its originalenvironment (eg. the natural environment if it is naturally occurring).For example, a naturally occurring nucleic acid or polypeptide presentin a living plant is not isolated, but the same nucleic acid orpolypeptide separated from some or all of the coexisting materials inthe natural system, is isolated. Such nucleic acids could be part of avector and/or such nucleic acids could be part of a composition, andstill be isolated in that such a vector or composition is not part ofits natural environment.

As used herein, the term “consensus contig” refers to a nucleotidesequence that is assembled from two or more constituent nucleotidesequences that share common or overlapping regions of sequence homology.For example, the nucleotide sequence of two or more nucleic acidfragments can be compared and aligned in order to identify common oroverlapping sequences. Where common or overlapping sequences existbetween two or more nucleic acid fragments, the sequences (and thustheir corresponding nucleic acid fragments) can be assembled into asingle contiguous nucleotide sequence.

The term “purified” means that the nucleic acid or polypeptide issubstantially free of other nucleic acids or polypeptides.

By “functionally active” in respect of a nucleic acid it is meant thatthe fragment or variant (such as an analogue, derivative or mutant) iscapable of modifying the tolerance of freezing and/or low temperaturestress in a plant. Such variants include naturally occurring allelicvariants and non-naturally occurring variants. Additions, deletions,substitutions and derivatizations of one or more of the nucleotides arecontemplated so long as the modifications do not result in loss offunctional activity of the fragment or variant. Preferably thefunctionally active fragment or variant has at least approximately 80%identity to the relevant part of the above mentioned sequence, morepreferably at least approximately 90% identity, most preferably at leastapproximately 95% identity. Such functionally active variants andfragments include, for example, those having nucleic acid changes whichresult in conservative amino acid substitutions of one or more residuesin the corresponding amino acid sequence. Preferably the fragment has asize of at least 30 nucleotides, more preferably at least 45nucleotides, most preferably at least 60 nucleotides.

By “functionally active” in respect of a polypeptide is meant that thefragment or variant has one or more of the biological properties of anIRIP or IRIP-like protein. Additions, deletions, substitutions andderivatizations of one or more of the amino acids are contemplated solong as the modifications do not result in loss of functional activityof the fragment or variant. Preferably the functionally active fragmentor variant has at least approximately 60% identity to the relevant partof the above mentioned sequence, more preferably at least approximately80% identity, most preferably at least approximately 90% identity. Suchfunctionally active variants and fragments include, for example, thosehaving conservative amino acid substitutions of one or more residues inthe corresponding amino acid sequence. Preferably the fragment has asize of at least 10 amino acids, more preferably at least 15 aminoacids, most preferably at least 20 amino acids.

The term “construct” as used herein refers to an artificially assembledor isolated nucleic acid molecule which includes the gene of interest.In general a construct may include the gene or genes of interest, amarker gene which in some cases can also be the gene of interest andappropriate regulatory sequences. It should be appreciated that theinclusion of regulatory sequences in a construct is optional, forexample, such sequences may not be required in situations where theregulatory sequences of a host cell are to be used. The term constructincludes vectors but should not be seen as being limited thereto.

The term “vector” as used herein encompasses both cloning and expressionvectors. Vectors are often recombinant molecules containing nucleic acidmolecules from several sources.

By “operatively linked” is meant that said regulatory element is capableof causing expression of said nucleic acid or nucleic acid fragment in aplant cell and said terminator is capable of terminating expression ofsaid nucleic acid or nucleic acid fragment in a plant cell. Preferably,said regulatory element is upstream of said nucleic acid or nucleic acidfragment and said terminator is downstream of said nucleic acid ornucleic acid fragment.

By “an effective amount” it is meant an amount sufficient to result inan identifiable phenotypic trait in said plant, or a plant, plant seedor other plant part derived therefrom. Such amounts can be readilydetermined by an appropriately skilled person, taking into account thetype of plant, the route of administration and other relevant factors.Such a person will readily be able to determine a suitable amount andmethod of administration. See, for example, Maniatis et al, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, the relevant disclosure of which is incorporated herein byreference.

It will also be understood that the term “comprises” (or its grammaticalvariants) as used in this specification is equivalent to the term“includes” and should not be taken as excluding the presence of otherelements or features.

Genes encoding other IRIP or IRIP-like proteins for modifying thetolerance of plants to freezing and/or low temperature stress, either ascDNAs or genomic DNAs, may be isolated directly by using all or aportion of the nucleic acids or nucleic acid fragments of the presentinvention as hybridisation probes to screen libraries from the desiredplant employing the methodology well known to those skilled in the art.Specific oligonucleotide probes based upon the nucleic acid sequences ofthe present invention may be designed and synthesized by methods knownin the art. Moreover, the entire sequences may be used directly tosynthesize DNA probes by methods known to the skilled artisan, such asrandom primer DNA labelling, nick translation, or end-labellingtechniques, or RNA probes using available in vitro transcriptionsystems. In addition, specific primers may be designed and used toamplify a part or all of the sequences of the present invention. Theresulting amplification products may be labelled directly duringamplification reactions or labelled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, short segments of the nucleic acids or nucleic acidfragments of the present invention may be used in protocols to amplifylonger nucleic acid fragments encoding homologous genes from DNA or RNA.For example, polymerase chain reaction may be performed on a library ofcloned nucleic acid fragments wherein the sequence of one primer isderived from the nucleic acid sequences of the present invention, andthe sequence of the other primer takes advantage of the presence of thepolyadenylic acid tracts to the 3′ end of the mRNA precursor encodingplant genes. Alternatively, the second primer sequence may be based uponsequences derived from the cloning vector. For example, those skilled inthe art can follow the RACE protocol [Frohman et al. (1988) Proc. Natl.Acad Sci. USA 85:8998, the entire disclosure of which is incorporatedherein by reference] to generate cDNAs by using PCR to amplify copies ofthe region between a single point in the transcript and the 3′ or 5′end. Using commercially available 3′ RACE and 5′ RACE systems (BRL),specific 3′ or 5′ cDNA fragments may be isolated [Ohara et al. (1989)Proc. Natl. Acad Sci USA 86:5673; Loh et al. (1989) Science 243:217, theentire disclosures of which are incorporated herein by reference].Products generated by the 3′ and 5′ RACE procedures may be combined togenerate full-length cDNAs.

In a further aspect of the present invention there is provided asubstantially purified or isolated IRIP or IRIP-like polypeptide from aDeschampsia species, preferably from Antarctic hair-grass, Deschampsiaantarctica; and functionally active fragments and variants thereof.

In a preferred embodiment of this aspect of the invention, thesubstantially purified or isolated polypeptide includes an amino acidsequence selected from the group consisting of sequences shown in FIGS.10, 13, 16, 19, 22 and 25 hereto; and functionally active fragments andvariants thereof.

In a still further aspect of the present invention there is provided asubstantially purified or isolated IRIP or IRIP-like polypeptide from aryegrass (Lolium) or fescue (Festuca) species; and functionally activefragments and variants thereof.

The ryegrass (Lolium) or fescue (Festuca) species may be of any suitabletype, including Italian or annual ryegrass, perennial ryegrass, tallfescue, meadow fescue and red fescue. Preferably the species is aryegrass, more preferably perennial ryegrass (L. perenne).

In a preferred embodiment of this aspect of the invention, thesubstantially purified or isolated polypeptide includes an amino acidsequence selected from the group consisting of sequences shown in FIGS.28 and 31 hereto; and functionally active fragments and variantsthereof.

The Applicant has found that the polypeptides of the present inventioninclude relatively few leucine rich repeat (LRR) motifs.

Preferably said LLR motifs from a Deschampsia species include theconsensus sequence:

LxLxxNxLTGxIPxxLGxLxxLxx (SEQ ID NO. 128)

or the consensus sequence:

LxLxxNxLSGxIPxxLGxLxxLxx (SEQ ID NO. 143)

Preferably said LRR motifs from a Lolium or Festuca species include theconsensus sequence:

LxLxxNxLTGxIPxxLGxLxxLxx (SEQ ID NO. 129)

or the consensus sequence:

LxLxxNxLSGxIPxxLGxLxxLxx (SEQ ID NO. 144)

Applicant has found that polypeptides of the present invention includingrelatively few LRR motifs, preferably 3 or fewer LRR motifs, morepreferably 1 or fewer LRR motifs, may be more efficient at modifyingtolerance of freezing and/or low temperature stress in a plant thannucleic acids or nucleic acid fragments having relatively more LRRmotifs, for example approximately 9 or more LRR motifs. Similarly, thenucleic acids or nucleic acid fragments encoding such polypeptides maybe more efficient at modifying tolerance of freezing and/or lowtemperature stress in a plant.

In a further embodiment of this aspect of the invention, there isprovided a polypeptide recombinantly produced from a nucleic acid ornucleic acid fragment according to the present invention. Techniques forrecombinantly producing polypeptides are known to those skilled in theart.

Availability of the nucleotide sequences of the present invention anddeduced amino acid sequences facilitates immunological screening of cDNAexpression libraries. Synthetic peptides representing portions of theinstant amino acid sequences may be synthesized. These peptides may beused to immunise animals to produce polyclonal or monoclonal antibodieswith specificity for peptides and/or proteins comprising the amino acidsequences. These antibodies may be then used to screen cDNA expressionlibraries to isolate full-length cDNA clones of interest.

A genotype is the genetic constitution of an individual or group.Variations in genotype are essential in commercial breeding programs, indetermining parentage, in diagnostics and fingerprinting, and the like.Genotypes can be readily described in terms of genetic markers. Agenetic marker identifies a specific region or locus in the genome. Themore genetic markers, the finer defined is the genotype. A geneticmarker becomes particularly useful when it is allelic between organismsbecause it then may serve to unambiguously identify an individual.Furthermore, a genetic marker becomes particularly useful when it isbased on nucleic acid sequence information that can unambiguouslyestablish a genotype of an individual and when the function encoded bysuch nucleic acid is known and is associated with a specific trait. Suchnucleic acids and/or nucleotide sequence information including singlenucleotide polymorphisms (SNP's), variations in single nucleotidesbetween allelic forms of such nucleotide sequence, can be used asperfect markers or candidate genes for the given trait.

Applicants have identified a number of SNPs of the nucleic acids ornucleic acid fragments of the present invention. These are indicated(marked with grey on the black background) in the figures that showmultiple alignments of nucleotide sequences of nucleic acid fragmentscontributing to consensus contig sequences. See for example, FIGS. 8,11, 14, 17, 20, 23, 26 and 29 hereto.

Accordingly, in a further aspect of the present invention, there isprovided a substantially purified or isolated nucleic acid or nucleicacid fragment including a single nucleotide polymorphism (SNP) from anucleic acid fragment shown in FIGS. 8, 9, 11, 12, 14, 15, 17, 18, 20,21, 23, 24, 26, 27, 29 and 30 hereto, or complements or sequencesantisense thereto.

In a still further aspect of the present invention there is provided amethod of isolating a nucleic acid or nucleic acid fragment of thepresent invention including a single nucleotide polymorphism (SNP), saidmethod including sequencing nucleic acid fragments from a nucleic acidlibrary.

The nucleic acid library may be of any suitable type and is preferably acDNA library.

The nucleic acid fragments may be isolated from recombinant plasmids ormay be amplified, for example using polymerase chain reaction.

The sequencing may be performed by techniques known to those skilled inthe art.

In a still further aspect of the present invention, there is provideduse of nucleic acids or nucleic acid fragments of the present inventionincluding SNP's, and/or nucleotide sequence information thereof, asmolecular genetic markers.

In a still further aspect of the present invention there is provided useof a nucleic acid or nucleic acid fragment according to the presentinvention, and/or nucleotide sequence information thereof, as amolecular genetic marker.

More particularly, nucleic acids or nucleic acid fragments according tothe present invention and/or nucleotide sequence information thereof maybe used as a molecular genetic marker for quantitative trait loci (QTL)tagging, QTL mapping, DNA fingerprinting and in marker assistedselection, particularly in grasses and cereals. Even more particularly,nucleic acids or nucleic acid fragments according to the presentinvention and/or nucleotide sequence information thereof may be used asmolecular genetic markers in grass and cereal improvement, e.g. taggingQTLs for tolerance to freezing and/or low temperature stress. Even moreparticularly, sequence information revealing SNPs in allelic variants ofthe nucleic acids or nucleic acid fragments of the present inventionand/or nucleotide sequence information thereof may be used as moleculargenetic markers for QTL tagging and mapping and in marker assistedselection, particularly in grasses and cereals.

In a still further aspect of the present invention there is provided aconstruct including a nucleic acid or nucleic acid fragment according tothe present invention. The construct may be a vector.

In a preferred embodiment of this aspect of the invention, the vectormay include a regulatory element such as a promoter, a nucleic acid ornucleic acid fragment according to the present invention and aterminator; said regulatory element, nucleic acid or nucleic acidfragment and terminator being operatively linked.

In a further preferred embodiment of this aspect of the invention, thevector may include a regulatory element according to the presentinvention, a further nucleic acid molecule and a terminator; saidregulatory element, further nucleic acid molecule and terminator beingoperatively linked.

In a still further preferred embodiment of this aspect of the invention,the vector may include a regulatory element according to the presentinvention, a nucleic acid or nucleic acid fragment according to thepresent invention and a terminator, said regulatory element, nucleicacid or nucleic acid fragment and terminator being operatively linked.

The vector may be of any suitable type and may be viral or non-viral.The vector may be an expression vector. Such vectors includechromosomal, non-chromosomal and synthetic nucleic acid sequences, eg.derivatives of plant viruses; bacterial plasmids; derivatives of the Tiplasmid from Agrobacterium tumefaciens, derivatives of the Ri plasmidfrom Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes;bacterial artificial chromosomes; binary bacterial artificialchromosomes; vectors derived from combinations of plasmids and phageDNA. However, any other vector may be used as long as it is replicable,or integrative or viable in the plant cell.

The regulatory element and terminator may be of any suitable type andmay be endogenous to the target plant cell or may be exogenous, providedthat they are functional in the target plant cell.

In another embodiment, the construct or vector may include more than onenucleic acid. The nucleic acids within the same construct or vector mayhave identical or differing sequences. In one preferred embodiment, theconstruct or vector has at least two nucleic acids encoding functionallysimilar enzymes. In a particularly preferred embodiment, each furthernucleic acid molecule has one or more upstream regulatory elements andone or more downstream terminators, although expression of more than onefurther nucleic acid molecule from an upstream regulatory element ortermination of more than one further nucleic acid molecule from adownstream terminator(s) is not precluded.

Preferably the regulatory element is a promoter. A variety of promoterswhich may be employed in the constructs and vectors of the presentinvention are well known to those skilled in the art. Factorsinfluencing the choice of promoter include the desired tissuespecificity of the vector, and whether constitutive or inducibleexpression is desired and the nature of the plant cell to be transformed(eg. monocotyledon or dicotyledon). Particularly suitable promotersinclude but are not limited to the constitutive Cauliflower Mosaic Virus35S (CaMV 35S) promoter and derivatives thereof, the maize Ubiquitinpromoter, the rice Actin promoter, and the tissue-specific Arabidopsissmall subunit (ASSU) promoter. Alternatively, the regulatory element maybe a regulatory element according to the present invention.

A variety of terminators which may be employed in the vectors andconstructs of the present invention are also well known to those skilledin the art. The terminator may be from the same gene as the promotersequence or a different gene. Particularly suitable terminators arepolyadenylation signals, such as the CaMV 35S polyA and otherterminators from the nopaline synthase (nos), the octopine synthase(ocs) and the rbcS genes.

The further nucleic acid molecule may be a sequence, for example a geneor fragment thereof, or sequence antisense thereto, which is capable ofmodifying plant response to freezing and/or low temperature stress. Itmay be a nucleic acid or nucleic acid fragment according to the presentinvention, but is not limited thereto.

The vector, in addition to the regulatory element, the nucleic acid ornucleic acid fragment of the present invention and the terminator, mayinclude further elements necessary for expression of the nucleic acid ornucleic acid fragment, in different combinations, for example vectorbackbone, origin of replication (ori), multiple cloning sites,recognition sites for recombination events, spacer sequences, enhancers,introns (such as the maize Ubiquitin (Ubi) intron), antibioticresistance genes and other selectable marker genes [such as the neomycinphosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph)gene, the phosphinothricin acetyltransferase (bar or pat) gene and thegentamycin acetyl transferase (aaacC1) gene], and reporter genes (suchas beta-glucuronidase (GUS) gene (gusA) and green fluorescent protein(gfp)]. The vector may also contain a ribosome binding site fortranslation initiation. The vector may also include appropriatesequences for amplifying expression.

As an alternative to use of a selectable marker gene to provide aphenotypic trait for selection of transformed host cells, the presenceof the construct or vector in transformed cells may be determined byother techniques well known in the art, such as PCR (polymerase chainreaction), Southern blot hybridisation analysis, histochemical GUSassays, visual examination including microscopic examination offluorescence emitted by gfp, northern and Western blot hybridisationanalyses.

Those skilled in the art will appreciate that the various components ofthe construct or vector are operatively linked, so as to result inexpression of said nucleic acid or nucleic acid fragment. Techniques foroperatively linking the components of the vector of the presentinvention are well known to those skilled in the art. Such techniquesinclude the use of linkers, such as synthetic linkers, for exampleincluding one or more restriction enzyme sites.

The constructs and vectors of the present invention may be incorporatedinto a variety of plants, including monocotyledons (such as grasses fromthe genera Deschampsia, Lolium, Festuca, Paspalum, Pennisetum, Panicumand other forage and turf grasses, corn, oat, sugarcane, wheat andbarley), dicotyledons (such as Arabidopsis, tobacco, white clover, redclover, subterranean clover, alfalfa, eucalyptus, potato, sugarbeet,canola, soybean, chickpea) and gymnosperms.

Techniques for incorporating the constructs and vectors of the presentinvention into plant cells (for example by transduction, transfection ortransformation) are well known to those skilled in the art. Suchtechniques include Agrobacterium-mediated introduction, electroporationto tissues, cells and protoplasts, protoplast fusion, injection intoreproductive organs, injection into immature embryos and high velocityprojectile introduction to cells, tissues, calli, immature and matureembryos. The choice of technique will depend largely on the type ofplant to be transformed.

Cells incorporating the constructs and vectors of the present inventionmay be selected, as described above, and then cultured in an appropriatemedium to regenerate transformed plants, using techniques well known inthe art. The culture conditions, such as temperature, pH and the like,will be apparent to the person skilled in the art. The resulting plantsmay be reproduced, either sexually or asexually, using methods wellknown in the art, to produce successive generations of transformedplants.

In a further aspect of the present invention there is provided a plantcell, plant, plant seed or other plant part, including, e.g. transformedwith, a construct or vector of the present invention.

The plant cell, plant, plant seed or other plant part may be from anysuitable species, including monocotyledons, dicotyledons andgymnosperms.

The present invention also provides a plant, plant seed or other plantpart, or a plant extract, derived from a plant cell of the presentinvention.

The present invention also provides a plant, plant seed or other plantpart, or a plant extract, derived from a plant of the present invention.

In a further aspect of the present invention there is provided a methodof modifying tolerance of freezing and/or low temperature stress in aplant, said method including introducing into said plant an effectiveamount of a nucleic acid or nucleic acid fragment, construct and/or avector according to the present invention.

Using the methods and materials of the present invention, the toleranceof freezing and/or low temperature stress in a plant may be increased ordecreased or otherwise modified. For example, the tolerance of freezingand/or low temperature stress may be increased or otherwise altered.They may be increased, for example, by incorporating additional copiesof a sense nucleic acid or nucleic acid fragment of the presentinvention. They may be decreased, for example, by incorporating anantisense nucleic acid or nucleic acid fragment of the presentinvention.

In a further aspect of the present invention there is provided apreparation for transforming a plant comprising at least one nucleicacid according to the present invention. The preparation may containvectors or other constructs to facilitate administration to and/ortransformation of the plant with the nucleic acid.

The present invention will now be more fully described with reference tothe accompanying Examples and drawings. It should be understood,however, that the description following is illustrative only and shouldnot be taken in any way as a restriction on the generality of theinvention described above.

In the Figures

FIG. 1. RI assay on total extracts of leaves from non-acclimated (grownat 22° C.) and cold acclimated (5° C.) D. antarctica. A, Initial icecrystal structure following snap freezing. B, Ice crystal structureafter 16 h incubation at −3° C. Capillary B contains extraction buffer;capillaries 1-7: 1000, 250, 62.5, 15.6, 3.91, 0.977 and 0.244 μg mL⁻¹respectively of total leaf protein. Extracts were either untreated orsubject to incubation at 95° C. for 5 min as indicated. Endpoint of RIactivity defined as the lowest protein concentration (μg mL⁻¹) at whichice crystal structure in B remains unchanged from that in A.

FIG. 2. A, Repeat structures of representative IRIP orthologues HvIRIP(SEQ ID NO. 126) and DaIRIPd (SEQ ID NO. 31). Four cysteine residuesconserved in LRR proteins, and predicted to form two disulphide bridgesare shown in bold, connected by lines to show predicted bridges. Ahighly conserved 9 amino acid motif including 3 of these cysteineresidues is underlined. Consensus sequences for plant LRRs (SEQ ID NOs.128 and 143) (Kobe and Kajava (2001) Curr Opin Struct Biol 11:725), andthe IRIP repeat (SEQ ID NO. 141) (this work) are shown in bold below thetandem repeat alignments, and identical residues highlighted by greyboxes. B, Sequence alignment of IRIP orthologues and a putative PSKRorthologue from Oryza sativa. Sequences include LpIRIP (Sidebottom, etal. (2000 Nature 406:256)) (SEQ ID NO. 124), predicted IRIP orthologueTaIRIP derived from assembly of T. aestivum sequences in the NCBI ESTdatabase (SEQ ID NO. 125), predicted HvIRIP derived from assembly of H.vulgare sequences in the NCBI EST database (SEQ ID NO. 126), predictedLmIRIP derived from assembly of L. multiflorum sequences in the NCBI ESTdatabase (SEQ ID NO. 142), and OsPSKR a putative PSKR orthologue fromOryza sativa (NP_(—)911036) (SEQ ID NO. 127). Sequences of the presentinvention are DaIRIPa (SEQ ID NO. 17), DaIRIPd (SEQ ID NO. 31) DaIRIPe.7(SEQ ID NO. 38), LpIRIPa (SEQ ID NO. 102) and LpIRIPb (SEQ ID NO. 120).Identical and conserved residues are highlighted by black and grey boxesrespectively. Four invariantly conserved cysteine residues are markedwith arrowheads. C, Schematic of domain organisation in IRIP orthologuesand OsPSKR. SP: signal peptide; 2× S—S; domain predicted to form 2disulphide bridges; LRRs numbered; Island/IsIa./Is.: island domain; IRIPrepeats unlabelled; TM: transmembrane domain. D, Phylogenetic tree ofIRIP orthologues and LRR proteins. Branch lengths are proportional tothe number of amino acid substitutions per site (indicated by scalebar). LRR proteins include accession number as suffix.

FIG. 3. Structural modelling of IRIPs. A, Theoretical structure ofDaIRIPa aligned along the prism face of ice (parallel to the a-axis). B,Theoretical structure of LpIRIPa aligned along the prism face of ice(parallel to the a-axis). Cysteine residues at positions 120 and 143have been modelled to participate in an additional disulphide bond,relative to DaIRIPa. C, Ribbon backbone diagram of DaIRIPa highlightingthe amino acid residue composition of the two putative ice bindingsurfaces, side “A” and side “B”. D, Cross-sectional view of ribbonbackbone of 2 β-roll loops of DaIRIPa showing positions of amino acidresidue side chains.

FIG. 4. Genomic organisation of IRIP genes. A, D. antarctica genomicSouthern probed with DaIRIPe. B, L. perenne polygenic genomic Southernprobed with LpIRIPa. C, L. perenne isogenic genomic Southern probed withLpIRIPa. 1: Undigested; 2: Sphl; 3: Kpnl; 4: HindIII; 5: BamHI.

FIG. 5. SNP-based genetic mapping. A, Distribution of locus-specificamplification primers, genomic amplicons and putative SNP loci acrossthe components of the LpIRIPa transcriptional unit. LpIRIP SNPs areindicated using the nomenclature na (number), indicating that the SNPwas identified between NA6 parental haplotypes, and the relevant basepair coordinate. SNPs derived from single gene copies that segregate inthe F₁ progeny are shown in bold, while SNPs that potentiallydiscriminate between paralogous gene copies are shown in normal text. B,Genetic map order in the upper part of the LG1 of the NA₆ parentalgenetic map, showing the LpIRIPNA476-detected SNP locus.

FIG. 6. Analysis of IRIP gene expression in response to temperature. A,Northern blot of RNA from D. antarctica leaves and roots grown at 22, 4,and −16° C., probed with DaIRIPe. B, Northern blot of RNA from L.perenne leaves and roots grown at 22 and 4° C., probed with LpIRIPa.

FIG. 7. RI assay on total extracts of E. coli expressing a putativeorthologue of histone H3.2 and DaIRIPe. A, Initial ice crystal structurefollowing snap freezing. B, Ice crystal structure after 16 h incubationat −3° C. Capillary EB contains extraction buffer; capillary BSA 1000 μgmL⁻¹ bovine serum albumin; capillary PC cold acclimated D. antarcticaleaf extract as positive control; capillaries 1-4: 400, 100, 25 and 6.25μg mL⁻¹ respectively of total extracts of E. coli.

FIG. 8. Nucleotide sequences of the nucleic acid fragments contributingto the consensus contig sequence DaIRIPa (SEQ ID NOs. 1-15)

FIG. 9. Consensus nucleotide sequence of DaIRIPa (SEQ ID NO. 16)

FIG. 10. Deduced amino acid sequence of DaIRIPa (SEQ ID NO. 17)

FIG. 11. Nucleotide sequences of the nucleic acid fragments contributingto the consensus contig sequence DaIRIPb (SEQ ID NOs. 18-20)

FIG. 12. Consensus nucleotide sequence of DaIRIPb (SEQ ID NO. 21)

FIG. 13. Deduced amino acid sequence of DaIRIPb (SEQ ID NO. 22)

FIG. 14. Nucleotide sequences of the nucleic acid fragments contributingto the consensus contig sequence DaIRIPd (SEQ ID NOs. 23-29)

FIG. 15. Consensus nucleotide sequence of DaIRIPd (SEQ ID NO. 30)

FIG. 16. Deduced amino acid sequence of DaIRIPd (SEQ ID NO. 31)

FIG. 17. Nucleotide sequences of the nucleic acid fragments contributingto the consensus contig sequence DaIRIPe7 (SEQ ID NOs. 32-36)

FIG. 18. Consensus nucleotide sequence of DaIRIPe7 (SEQ ID NO. 37)

FIG. 19. Deduced amino acid sequence of DaIRIPe7 (SEQ ID NO. 38)

FIG. 20. Nucleotide sequences of the nucleic acid fragments contributingto the consensus contig sequence DaIRIPe8 (SEQ ID NOs. 39-44)

FIG. 21. Nucleotide sequence of DaIRIPe8 (SEQ ID NO. 45)

FIG. 22. Deduced amino acid sequence of DaIRIPe8 (SEQ ID NO. 46)

FIG. 23. Nucleotide sequences of the nucleic acid fragments contributingto the consensus contig sequence DaIRIPf (SEQ ID NOs. 47-52)

FIG. 24. Consensus nucleotide sequence of DaIRIPf (SEQ ID NO. 53)

FIG. 25. Deduced amino acid sequence of DaIRIPf (SEQ ID NO. 54)

FIG. 26. Nucleotide sequences of the nucleic acid fragments contributingto the consensus contig sequence LpIRIPa (SEQ ID NOs. 55-100)

FIG. 27. Consensus nucleotide sequence of LpIRIPa (SEQ ID NO. 101)

FIG. 28. Deduced amino acid sequence of LpIRIPa (SEQ ID NO. 102)

FIG. 29. Nucleotide sequences of the nucleic acid fragments contributingto the consensus contig sequence LpIRIPb (SEQ ID NOs. 103-118)

FIG. 30. Consensus nucleotide sequence of LpIRIPb (SEQ ID NO. 119)

FIG. 31. Deduced amino acid sequence of LpIRIPb (SEQ ID NO. 120)

FIG. 32. Nucleotide sequence of promoter region of DaIRIPa extending tothe initiating ATG (underlined) (SEQ ID NO. 121)

FIG. 33. Nucleotide sequence of promoter region of DaIRIPd extending tothe initiating ATG (underlined) (SEQ ID NO. 122)

FIG. 34. Nucleotide sequence of promoter region of LpIRIPa extending tothe initiating ATG (underlined) (SEQ ID NO. 123)

FIG. 35. Plasmid map of vector used for DaIRIPa gain of functionbiolistic transformation of wheat.

FIG. 36. Plasmid map of vector used for DaIRIPd gain of functionbiolistic transformation of wheat.

FIG. 37. Plasmid map of vector used for DaIRIPe7 gain of functionbiolistic transformation of wheat.

FIG. 38. Plasmid map of vector used for DaIRIPe8 gain of functionbiolistic transformation of wheat.

FIG. 39. Plasmid map of vector used for LpIRIPa gain of functionbiolistic transformation of ryegrass.

FIG. 40. Plasmid map of vector used for LpIRIPb gain of functionbiolistic transformation of ryegrass.

FIG. 41. Plasmid map of vector used for LpIRIPa loss of functionbiolistic transformation of ryegrass.

FIG. 42. Plasmid map of vector used for LpIRIPb loss of functionbiolistic transformation of ryegrass.

FIG. 43. Plasmid map of vector used for DaIRIPa gain of functionAgrobacterium mediated transformation of wheat and barley.

FIG. 44. Plasmid map of vector used for DaIRIPd gain of functionAgrobacterium mediated transformation of wheat and barley.

FIG. 45. Plasmid map of vector used for DaIRIPe7 gain of functionAgrobacterium mediated transformation of wheat and barley.

FIG. 46. Plasmid map of vector used for DaIRIPe8 gain of functionAgrobacterium mediated transformation of wheat and barley.

FIG. 47. Plasmid map of vector used for DaIRIPa gain of functionAgrobacterium mediated transformation of Arabidopsis.

FIG. 48. Plasmid map of vector used for DaIRIPd gain of functionAgrobacterium mediated transformation of Arabidopsis.

FIG. 49. Plasmid map of vector used for DaIRIPe7 gain of functionAgrobacterium mediated transformation of Arabidopsis.

FIG. 50. Plasmid map of vector used for DaIRIPe8 gain of functionAgrobacterium mediated transformation of Arabidopsis.

FIG. 51. Plasmid map of vector used for LpIRIPa gain of functionAgrobacterium mediated transformation of Arabidopsis.

FIG. 52. Plasmid map of vector used for LpIRIPb gain of functionAgrobacterium mediated transformation of Arabidopsis.

FIG. 53. Plasmid map of vector used for DaIRIPd promoter::GUS reportergene Agrobacterium mediated transformation of Arabidopsis.

EXAMPLE 1

Materials and Methods

Plant Propagation, Stress Treatments, Extraction of RI Activity, DNA andRNA

D. antarctica material was collected in the vicinity of Jubany stationon King George Island (62°14′S 58°40′W). Plants were germinated fromseeds in the soil seed bank and thereafter were propagated vegetatively.Lolium perenne plants were of cultivar Impact. Doubled haploid L.perenne plants, where isolate DH297 of cultivar Verna.

Individual plants were grown in soil at the indicated temperatures undera 16/8 h light/dark regime and photosynthetic photon flux intensity of400 μmol m⁻² s⁻¹ in Enconair (Winnipeg, Canada) GC-20 plant growthchambers. Plants were cold acclimated by growth at 5° C. for 2 weeks.Plants were divided into aerial and subterranean parts and snap frozenin liquid N₂.

Total cellular extracts were prepared after (Doucet et al (2000)Cryobiology 40:218) by grinding plant material under liquid N₂ andresuspending the powder in 2 mL g⁻¹ of freshly prepared extractionbuffer (50 mM Tris pH 7.4, 20 mM ascorbate, 10 mM EDTA). The extract wasfiltered through miracloth (Calbiochem, La Jolla, USA). Apoplasticextracts were based on the method of Chun et al (1998) Euphytica102:219. Leaf material was vacuum infiltrated in extraction buffer for30 min, excess liquid removed from the leaves, and extracts collected bycentrifugation at 800 g for 30 min. All extracts were aliquoted, frozenin liquid N₂, and stored at −80° C.

RNA and DNA were extracted using RNeasy and DNeasy Plant Mini kits(QIAGEN, Germany) respectively.

Ice Recrystallisation Inhibition (RI) Assays

Extracts were thawed and insoluble material pelleted by centrifugationat 16,060 g for 5 min. Supernatants were collected and for totalcellular extracts protein content quantified using the Bio-Rad proteinassay (Bio-Rad, Mississauga, ON, Canada), according to themanufacturer's instructions. Unless otherwise stated all extracts wereincubated at 95° C. for 5 min. The supernatants from both heat treatedand untreated extracts were collected following centrifugation at 16,060g for 2 min. Serial 2 or 4-fold dilutions into extraction buffer wereprepared.

The capillary method for the RI assay was modified from that of Tomczaket al (2003) Biochem. Biophys. Res. Commun. 311:1041. Briefly, extractswere loaded into 10 μl glass capillaries (Drummond Scientific, Broomall,Pa., USA), heat sealed and arrayed on a glass slide secured withadhesive tape. Extraction buffer, and BSA (Bio-Rad, Mississauga, ON,Canada) dissolved in extraction buffer to a final concentration of 1000μg mL⁻¹ were included as negative controls. The capillary array wassnap-frozen in an ethanol/dry ice bath and immersed in a reservoir ofmotor vehicle coolant diluted to a final concentration of 10%monoethylene glycol, atop a jacketed stage through which the samesolution at −3° C. was circulated using a refrigerated water bath(PolyScience Model 910, Niles, Ill., USA).

Samples were scored after overnight (˜16 h) incubation at −3° C. Theendpoint of RI activity in total leaf extracts is defined as the lowestprotein concentration (μg mL⁻¹) at which ice crystal structure followingincubation at −3° C. for 16 h, remained unchanged from that initiallyinduced by snap freezing. For apoplastic extracts, because of the lowyields of protein from non-acclimated plants, the endpoint of RIactivity was expressed as the equivalent wet weight of starting plantmaterial per volume of extract.

Digital images were captured with a Leica DFC 300 F camera mounted on aLeica MZFL III stereoscopic zoom microscope using Leica FireCam software(Leica, Heerbrugg, Switzerland). Polarising light filters mountedperpendicularly on the microscope objective lens and beneath the stageenhanced visualisation of ice crystal morphology.

Preparation of cDNA Libraries, Isolation and Sequencing of cDNAs Codingfor IRIPs from Antarctic Hair-Grass, Deschampsia antarctica.

cDNA libraries representing mRNAs from various organs and tissues fromAntarctic hair-grass, Deschampsia antarctica were prepared. Thecharacteristics of the libraries are described below (Table 1).

TABLE 1 cDNA libraries from Antarctic hair-grass, Deschampsiaantarctica. Library Organ/Tissue 05Da Aerial parts grown at 4° C. 08DaRoots grown at −15° C. 09Da Roots transferred from −15° C. to 25° C. for24 h 10Da Aerial parts transferred from −15° C. to 25° C. for 24 h 11DaAerial parts grown at −15° C. 12Da Roots grown at −15° C. 15Da Rootsgrown at 4° C. 16Da Aerial parts grown at 4° C. 17Da Roots transferredfrom 25° C. to 0° C. for 48 h 18Da Aerial parts transferred from −15° C.to 0° C. for 48 h 19Da Aerial parts transferred from 25° C. to 0° C. for48 h, then to −15° C. for 48 h 20Da Aerial parts grown at −15° C. 21DaAerial parts grown at 4° C. 22Da Roots grown at −15° C. 23Da Roots grownat 4° C.

The cDNA libraries may be prepared by any of many methods available. Forexample, total RNA may be isolated using the Trizol method (Gibco-BRL,USA) or the RNeasy Plant Mini kit (Qiagen, Germany), following themanufacturers' instructions. cDNAs may be generated using the SMART PCRcDNA synthesis kit (Clontech, USA), cDNAs may be amplified by longdistance polymerase chain reaction using the Advantage 2 PCR Enzymesystem (Clontech, USA), cDNAs may be cleaned using the GeneClean spincolumn (Bio 101, USA), tailed and size fractionated, according to theprotocol provided by Clontech. The cDNAs may be introduced into thepGEM-T Easy Vector system 1 (Promega, USA) according to the protocolprovided by Promega. The cDNAs in the pGEM-T Easy plasmid vector aretransfected into Escherichia coli Epicurian coli XL10-Gold ultracompetent cells (Stratagene, USA) according to the protocol provided byStratagene.

Alternatively, the cDNAs may be introduced into plasmid vectors forfirst preparing the cDNA libraries in Uni-ZAP XR vectors according tothe manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif., USA). The Uni-ZAP XR libraries are converted into plasmidlibraries according to the protocol provided by Stratagene. Uponconversion, cDNA inserts will be contained in the plasmid vectorpBluescript. In addition, the cDNAs may be introduced directly intoprecut pBluescript II SK(+) vectors (Stratagene) using T4 DNA ligase(New England Biolabs), followed by transfection into E. coli DH10B cellsaccording to the manufacturer's protocol (GIBCO BRL Products). cDNAclones encoding putative IRIP orthologues from D. antarctica came from 5libraries derived from either shoots or roots grown at 4° C. or −15° C.,and from shoots transferred from −15° C. to 25° C. for 24 hours. One ofthese variants (DaIRIPd) was isolated from both root and shootlibraries, whilst the other four forms were derived from shoot librariesonly.

Molecular Cloning of Genomic Sequences

All but one of D. antarctica cDNAs encoding IRIP orthologues (DaIRIPa),encoded N-terminally truncated hypothetical IRIP forms. Therefore fulllength genomic sequences where isolated using the GenomeWalker kit (BDBiosciences, Palo Alto, USA) and nested gene specific primers. The 3′UTR primers for DaIRIPd were: 5′ GACATCGCGATTGGTCCCACCAAGTG 3′ (SEQ IDNO. 130), and 5′ GCATCCTGCACGGACATATCATTA 3′ (SEQ ID NO. 131); andDaIRIPe: 5′ GTTACATAAGACGATTGGCCCCACCAAG 3′ (SEQ ID NO. 132), and 5′CAATCCACTCACTGATCATTAACCACC 3′ (SEQ ID NO. 133). For the isolation ofLpIRIPa nested primers 5′ GATGCTATATCCACGAAGTTACAT 3′ (SEQ ID NO. 134),and 5′ ATTGGCCCCACCAAGTGA 3′ (SEQ ID NO. 135) complementary to conservedregions within the 3′ UTR of the D. antarctica IRIP forms were employed.LpIRIPa was also obtained from the L. perenne North African₆×Aurora₆genetic mapping population (see below). PCR products were purified usingQIAquick gel extraction kit (QIAGEN, Germany) and molecularly clonedinto pGEM-T Easy as above.

DNA Template Preparation, Sequencing and Analysis

Templates for sequencing of cDNA and genomic clones plasmid DNA waspurified using a QIAprep spin miniprep kit (QIAGEN, Germany). Sequencingreactions primed with a modified SMART primer (5′AAGCAGTGGTAACAACGCAGAGTGGG 3′) (SEQ ID NO. 136), M13F or M13R primerswere performed either using BigDye Terminators or ET Terminators, andthe reaction products resolved on an ABI Prism 3700, or 3730xl DNAAnalyser (PE Biosystems, Foster City, USA), or a MegaBACE 4000 (AmershamBiosciences, UK) respectively. Sequence files were used as queries forBLASTX, BLASTN and TBLASTN Altschul et al 1987 Nucleic Acids Res.25:3389 searches of the SWISS-PROT, GenBank Main and GenBank ESTdatabases respectively. BLASTX searches of the NCBI database ofGenBank+EMBL+DDBJ sequences from EST divisions with full length IRIPgene sequences reveal the presence of many IRIP gene related sequencesin grasses including Lolium multiflorum (Italian rye grass), Leymuschinensis, Puccinellia tenuiflora, and from in-house EST data Agrostistenuis. We have also identified in the NCBI EST database orthologoussequences in the cereals Hordeum vulgare (barley), S. cereale (winterrye), and Triticum monococcum, T. turgidum and T. aestivum (diploid,tetraploid and hexaploid forms of wheat), frequently associated withcDNA libraries derived from cold-stressed or vernalised material. We arenot yet aware of any species of the Pooideae without IRIP orthologues.EST sequences were also assembled to derive representative IRIPorthologues. Sequence assembly was performed using Sequencher (GeneCodes, Ann Arbor, Mich., USA). Potential signal sequences wereidentified by analysis with SignaIP (www.cbs.dtu.dk/services/SignaIP/).Subcellular localisation was predicted using TMHMM(http://www.cbs.dtu.dk/services/TMHMM/) and PSORT (psort.nibb.ac.jp).Phylogenic analysis was performed using Vector NTI (Invitrogen,Carlsbad, Calif., USA) using the Align X algorithm with defaultparameters.

Structural Modelling

Homology modelling was performed using Schrödinger molecular modellingsoftware (version macromodel 8.6, Portland, Oreg., USA) using the Primehomology modelling module. A homology model of the DaIRIPa and LpIRIPaproteins was build using the N terminus of the crystal structure ofPhaseolus vulgaris polygalacturonase-inhibiting protein (PGIP) (pdbentry: 1OGQ) and a right-handed version of a theoretical Lolium perenneAFP model (Kuiper et al (2001) Biophys. J. 81:3560) as templates. Themodel was geometrically optimised with distance constraints holdingoptimal hydrogen bond distances between beta sheet regions for 10,000iterations using an OPLS2001 forcefield and Generalised Born (GB)solvation. This was followed by an additional 5000 iterationsminimisation without constraints applied. Images were generated usingSwiss Pdb Viewer and Pov-Ray3.5.

Southern and Northern Analysis

For Southerns 10 μg of DNA was digested with restriction enzymes,separated on a 1% agarose TAE gel and stained with ethidium bromidebefore transfer to Hybond-N membrane (Amersham Biosciences). ForNortherns 10 μg of RNA was separated on a 1% agarose formaldehyde gel,transferred to Hybond-N membrane (Amersham Biosciences) and stained withmethylene blue. Isolated fragments of DaIRIPe7 and LpIRIPa wereradio-labelled with α-³²P-dCTP using the Rediprime II Random PrimeLabelling System (Amersham Biosciences) and purified with MicroSpin™S-400 HR Columns (Amersham Biosciences) according to the manufacturer'sinstructions. The blotted membranes were hybridised with radio-labelledprobe as described by Maniatis et al, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor. Hybridisationpatterns were visualised and quantified on a Typhoon 8600 Variable ModeImager (Amersham Biosciences) according to the manufacturersinstructions.

SNP-Based Genetic Mapping

The perennial ryegrass population used for SNP discovery and geneticmapping was an F₁ progeny set derived from a reciprocal pair-crossbetween the heterozygous parental genotypes North African₆ (NA_(B)) andAurora₆ (AU₆) (Faville et al., Theor Appl Genet. in press).

The procedure for targeted in vitro SNP discovery in perennial ryegrasswas described by Forster et al. (2004) Molecular marker-based geneticanalysis of pasture and turf grasses, in: Molecular Breeding of Forageand Turf, Hopkins et al (eds) Kluwer Academic Press pp 197-239. Thepartial cDNA sequence of the IRIP reported from L. perenne (Sidebottomet al (2000) Nature 406:256), along with the sequence of LpIRIPa, wereassembled into a single contig and locus amplification primers (LAPs)were designed to generate 3 amplicons located at various positionswithin the gene unit (FIG. 5A), to cover the 5′-untranslated region(UTR), single exon and 3′-UTR regions. Genomic amplicons were generatedusing standard PCR conditions from each parental genotype of theF₁(NA₆×AU₆) population. PCR fragments were cloned using the TOPO TAsystem (Invitrogen K4575-40) and DNA sequences were derived throughcycle-sequencing. Sequences were assembled in contigs using theSequencher ver. 4.1.4 application (Gene Codes) and putative SNPs wereidentified. A total of 1072 bp from the 1495 bp gene length wasrepresented by high quality sequence. In all 26 SNPs were identifiedfollowing alignment of sequences from the parental genotypes, at anaverage incidence of 1 per 40 bp, which is higher than the globalfrequency of 1 per 60 bp observed through resequencing of 87 genes andc. 76 kb of perennial ryegrass genomic DNA (N.O.I. Cogan, unpublisheddata). This high SNP frequency, along with the observation of more thantwo haplotype structures in the NA₆ parent, suggests that paralogous DNAsequences may have been clustered, due to cross-amplification betweengene copies. Polymorphic SNP loci were validated using the singlenucleotide primer extension (SNuPe) assay system followed by capillaryelectrophoresis on the MegaBACE1000 platform (Amersham Biosciences).

Genotypic variation for the SNP locus LpIRIPNA476 was determined using asub-set (96) genotypes of the F₁(NA₆×AU₆) population. The correspondinggenomic locus was integrated within the framework of the existinggenetic map as previously described (Faville et al., Theor Appl Genet.in press). Comparative genomics analysis of IRIP genes was performedusing the wEST SQL database in the GrainGenes resource(http://wheat.pw.usda.gov/wEST/). The nucleotide sequences were used forBLASTN and TBLASTX analysis in the GrainGenes BLAST page with the searchrestricted to ESTs that have been assigned to wheat deletion bins Qi etal (2003) Functional and Integrative Genomics 3:39) The highest matchingESTs were then used to detect the relevant deletion bins using theMapped Loci query function in wEST SQL.

Genetic map information for the LpIRIPb gene was obtained from thesecond generation perennial ryegrass genetic mapping population derivedfrom a pair-cross between the genotypes North African₆ (NA₆) and Aurora₆(AU₆). LpIRIPa specific locus amplification primers (LAPs) were employedto generate 3 genomic amplicons (FIG. 5A) from each parental genotype.Sequence assembly and analysis revealed 2 distinct LpIRIP paraloguesLpIRIP a and b, and a total of 26 SNPs. An assayed SNP locus in LpIRIPashowing an AB×BB segregation structure produced only AB-type F₁ progeny(data not shown), which is diagnostic of multiple gene structure. Atotal of 8 SNP loci in LpIRIPb showed structures consistent with asingle gene copy, but of these 6 showed AA×BB patterns, and could not begenetically mapped in the F₁(NA₆×AU₆) sib-ship. Of the two locirevealing polymorphism within the NA₆ parental genotype (LpIRIPNA476 andLpIRIPNA694) (FIG. 5A), LpIRIPNA476 was used to genotype the mappingpopulation. The corresponding genomic locus was located on NA₆ LG1 inthe terminal location c. 7 cM from locus xLpesi3f (FIG. 5B).

Expression Profiling: RT-PCR?

Expression in E. coli

The sequence encoding DaIRIPe and a putative orthologue of histone H3.2was PCR adapted with the primer pairs 5′CAGCTTGGATCCATGGCGAACTGCTGTCTGCTA 3′ (SEQ ID NO. 137) and 5′ACTCACAAGCTTAACCTCCTGTCACGACTTTGT 3′ (SEQ ID NO. 138); and 5′AGGAGAGGATCCATGGCGCGTACCAAACAGACC 3′ (SEQ ID NO. 139) and 5′TAATTGAAGCTTTTAGGCGCGTTCGCCACGGAT 3′ (SEQ ID NO. 140) respectively. Theywere molecularly cloned into BamHI and HindIII restricted pQE-30(QIAGEN, Germany) and transformed into M15[pREP4]. To induce expressiona culture was grown in the presence of ampicillin and kanamycin tomid-log phase, where upon IPTG to 1 mM was added and incubationcontinued for a further 4 h. Cells were harvested, resuspended in 1 mlextraction buffer and lysed by sonication. The lysate was incubated at95° C. for 10 min., spun at 16,060 g for 5 min, the supernatantaliquoted, frozen in liquid N₂, and stored at −80° C.

EXAMPLE 2

Results

RI Activity in D. antarctica and L. perenne is Induced by ColdAcclimation and Present in the Apoplasm

RI assays reveal that D. antarctica has activity, induced by coldacclimation, to inhibit further growth of ice crystals followingfreezing. Given that total leaf extracts from plants grown at 22° C.containing 1000 μg mL⁻¹ of protein possess no RI activity (FIG. 1)transfer of plants to 5° C. for 2 weeks induces RI activity by greaterthan 64 fold. Furthermore this activity is unaffected by incubation at95° C. for 5 min (FIG. 1). RI activity is also induced more than 8 foldin the roots of D. antarctica in response to cold acclimation (Table 2).Similarly RI activity in L. perenne is below the threshold of detectionin the leaves and roots of non-acclimated plants but is induced inexcess of 16 and 4 fold respectively following cold acclimation (Table2).

TABLE 2 RI activity^(a) in leaves and roots of non-acclimated (grown at22° C.) and cold acclimated (5° C.) D. antarctica and L. perenne. LeavesRoots 22° C. 5° C. 22° C. 5° C. D. antarctica ND^(b) 15.6 ND^(b) 100 L.perenne ND^(b) 62.5 ND^(b) 200 ^(a)Expressed as lowest concentration oftotal protein extract (μg mL⁻¹) at which activity retained. ^(b)Noactivity detectable at 1000 and 800 μg mL⁻¹ for leaves and rootsrespectively.

Moreover RI activity is present in the extracellular spaces of D.antarctica and L. perenne. Leaf apoplastic extracts from plants of bothspecies grown at 22° C. possess no RI activity whilst activity isinduced in response to acclimation at least 73 fold in D. antarctica and1.7 fold in L. perenne (Table 3). These correspond to apoplastic proteinconcentrations, in cold acclimated plants, of 0.31 and 14 μg mL⁻¹respectively.

TABLE 3 RI activity^(a) in apoplastic extracts of leaves fromnon-acclimated (grown at 22° C.) and cold acclimated (5° C.) D.antarctica and L. perenne. 22° C. 5° C. D. antarctica ND^(b) 89.1 L.perenne ND^(b) 3,830 ^(a)Expressed as the lowest equivalent wet weightof starting plant material per volume of extract (mg mL⁻¹) at whichactivity retained. ^(b)No activity detectable at 6,550 and 6,590 mg mL⁻¹for D. antarctica and L. perenne respectively.

Therefore activity to inhibit the consolidation of ice crystals byrecrystallisation is induced in response to cold acclimation in bothleaves and roots of D. antarctica, and to a significantly lesser extentin L. perenne. Moreover a significant proportion of this RI activity,particularly in D. antarctica, is localised to apoplastic spaces.

IRIP Orthologues from D. antarctica are Predicted to be SecretedProteins and Contain Two Types of Repeat Motif.

Full length clones of the putative IRIP orthologues DaIRIPa, andDaIRIPd, e7 and e8 were obtained from D. antarctica cDNA and genomicresources respectively. Two genomic clones encoding putative IRIPparalogues LpIRIP a and b were also obtained from L. perenne. Inaddition many IRIP related sequences have been identified in ESTcollections from other cereals and grasses. The repeat structures of thelongest IRIP orthologue, HvIRIP from Hordeum vulgare (barley), and theshortest, DaIRIPd, are shown in FIG. 2A. In all predicted IRIPorthologues (FIG. 2B, C), the C-terminal approximate 120 residuesconsist entirely of 16 tandem repeats of a degenerate 7-8 amino acidresidue motif (the “IRIP repeat”) (FIG. 2A). The consensus sequence forthe IRIP repeat is SNNTWSG (SEQ ID NO. 141), with the glycine residuebeing most conserved (91.9% identity) across all forms. A multiplesequence alignment (FIG. 2B) indicates that relative to IRIP forms inother species IRIPs from L. perenne lack the 14th IRIP repeat but havean additional highly degenerate repeat immediately N-terminal to theusual start position of the IRIP domain (FIG. 2B, C).

Database sequence similarity searches with IRIP sequences reveal thatthe region N-terminal to the IRIP domain is related to proteins withleucine rich repeat (LRR) motifs. Most closely related is a putativeOryza sativa orthologue of a phytohormone receptor, the phytosulfokinereceptor (PSKR) (NP_(—)911036). Regions of significant sequencesimilarity with IRIPs extend in a discontinuous fashion through thefirst 17 LRRs of the putative PSKR orthologue and approximately 22residues into a 36 amino acid residue “island” domain (Li and Chory(1997) Cell 90:929), where similarity ceases with the advent of the IRIPdomain (FIG. 2B, C). With reference to the organisation of LRRs in theO. sativa PSKR orthologue IRIPs lack between 8 and 16 of the 17 LRRs(FIG. 2B, C).

Phylogenetic analysis on the sequences of IRIP orthologues outside ofthe IRIP domain, together with representative LRR containing proteinsreveal that IRIPs fall into a highly robust and distinct clade (FIG.2D). The sister group to the IRIP clade includes PSKR orthologues andcontains LRR receptor-like kinases (LRR-RLKs) of both monocot and dicotorigin (FIG. 2D). The most distant clade in this analysis includespolygalacturonase-inhibiting protein (PGIP) orthologues, and a LRRcontaining AFP from D. carota (FIG. 2D).

Immediately N-terminal to the LRRs in the predicted IRIPs is a highlyconserved 10 amino acid residue motif CCXWEGVXCD (SEQ ID NO. 145)containing 3 invariant cysteine residues (FIG. 2A, B). An additionalinvariant cysteine residue occurs a further 31-32 residues proximal tothe N-terminus (FIG. 2A, B). The corresponding cysteine residues havebeen shown to form 2 conformationally critical disulphide bridges in thestructural determination of the LRR-RLK PGIP of Phaseolus vulgaris (DiMatteo et at (2003) Proc. Natl. Acad Sci. USA 100:10124). It is surmisedbased on their conservation in all IRIP forms that the orthologousresidues also participate in structurally important disulphide bonds.

At the N-terminus of all IRIP orthologues is a 20 or 21 amino acidresidue region predicted to function as a signal peptide, with acleavage site between conserved alanine, and threonine or valineresidues (FIG. 2B). Consistent with this the mature versions of all fulllength IRIP forms are predicted to be extracellularly localised. Thus itis likely that IRIPs, are predominantly apoplastic.

Structural Modelling of IRIPs Predict Conformations that are LatticeMatched to Ice Surfaces

Three-dimensional structures of DaIRIPa and LpIRIPa (FIG. 3) wereconstructed by comparative homology modelling. The structural model ofDaIRIPa has three main regions, the double disulphide bonded N-terminaldomain, one LRR loop and the IRIP domain (FIG. 3A). LpIRIPa is similarto the DaIRIPe in overall structure except for its three LRRs, andadditional predicted disulphide bond (FIG. 3B).

The extended β-roll structure of the IRIP domain is predicted to formtwo surfaces complementary to the prism face of ice, on alternate sidesof the domain. Adjacent parallel β -loops are spaced approximately 4.5 Åapart (FIG. 3A, B), whilst threonine and other solvent-accessibleresidues are arrayed in two ranks on the β-strand faces, spaced 2residues, or approximately 7.4 Å apart (FIG. 3C). This almost exactlymatches the prism ice surface that has repeating structures 4.5 Åparallel to the a-axis, and 7.35 Å parallel to the c-axis of ice. Theputative ice-binding surfaces are stabilised by valine residues thatallow tight regular hydrophobic packing of the central core of theβ-roll region, and by asparagine residues that participate in hydrogenbonds between adjacent β-strands (FIG. 3D). The highly conserved glycineresidues in the IRIP repeat are structurally important as they form theturns between the upper and lower β-strand faces of the ice-bindingβ-roll.

Although the conformation of the LRR regions of the DaIRIPa and LpIRIPamodels recapitulate the right-handed β-roll of the IRIP repeat they donot engender a flat β-sheet roll. On one side of the β-roll adjacentparallel β-loops are spaced approximately 4.5 Å apart, but on the other,adjacently α-helical strands cannot pack as closely (FIG. 3A, B).Therefore with each iteration of the LRR the β-roll structure becomesincreasingly curved, displaying a concave β-sheet region. As aconsequence relative to DaIRIPa the predicted LpIRIPa structure withthree LRRs appears to display a less than optimal surface lattice matchto the ice prism face (FIG. 3B).

Genomic Organisation of IRIP Related Sequences in D. antarctica and L.perenne

Consistent with the multiple IRIP gene variants isolated from them, thegenomes of D. antarctica and L. perenne both appear to harbour multipleIRIP-related sequences. Up to 5 hybridising bands are detected byinterrogation of a Southern blot of D. antarctica genomic DNA withDaIRIPe (FIG. 4A). The occurrence of as few as 2 hybridising bands(tracks 3 and 5, FIG. 4A), is evidence that IRIP related sequences maybe physically linked on 2 genomic fragments totalling 20 kbp. At least 4LpIRIPa sequence related restriction fragments are detected in a samplegenome from a heterogeneous breeding population of L. perenne (FIG. 4B).The isogenic genomic DNA from a doubled haploid plant, however, exhibitsonly one strongly hybridising band, with a background of less intensebands (FIG. 4C).

SNP-Based Genetic Mapping of LpIRIPb

The genetic map position of LpIRIPb was determined using singlenucleotide polymorphism (SNP). Genetic map information for the LpIRIPbgene was obtained from the second generation perennial ryegrass geneticmapping population derived from a pair-cross between the genotypes NorthAfrican₆ (NA₆) and Aurora₆ (AU₆). A total of 8 SNP loci in LpIRIPb (FIG.5A) showed structures consistent with a single gene copy, but of these 6showed AA×BB patterns, and could not be genetically mapped in theF₁(NA₆×AU₆) sib-ship. Of the two loci revealing polymorphism within theNA₆ parental genotype (LpIRIPNA476 and LpIRIPNA694) (FIG. 5A),LpIRIPNA476 was used to genotype the mapping population. Thecorresponding genomic locus was located on NA₆ LG1 in the terminallocation c. 7 cM from locus xLpesi3f (FIG. 5B).

The DNA sequence from LpIRIPb was used to detect putatively orthologouswheat ESTs that had been assigned to the wheat deletion map (Endo andGill (1996) Journal of Heredity 87:95; Qi et al (2003) Functional andIntegrative Genomics 3:39). The three highest matching ESTs based onTBLASTX analysis were all assigned to deletion bins on chromosomes 4AL,5BL and 5DL (BE48991: E=7×10⁻⁷¹; BF200590: E=5×10⁻⁵⁸; BG314423:E=2×10⁻⁴¹). The next lowest matching wheat EST (BG607348: E=2×10⁻⁴⁰)detected a deletion bin on chromosome 1BL, as well as 5BL.

Expression Analysis of LpIRIPs and DaIRIPs

The modulation of steady state levels of IRIP gene transcripts inresponse to temperature in D. antarctica and L. perenne, wereinvestigated. A Northern blot comprising RNA samples extracted from theroots and leaves of D. antarctica plants grown at 22° C., 4° C. and −16°C. probed with full length DaIRIPe7 detects appreciable levels oftranscript only in leaves of cold-acclimated plants (FIG. 6A).Quantitative analysis reveal that transfer of plants to 4° C. for 2weeks increases steady state IRIP mRNA levels approximately 47 foldrelative to those grown at 22° C. By contrast LpIRIPa transcript levelsin L perenne are below the threshold level of detection in leaves, butelevated approximately 4 fold in the roots of cold-acclimated plantsrelative to those grown at 22° C. (FIG. 6B). Thus steady state levels ofIRIP transcripts are greatly elaborated in leaves of D. antarctica inresponse to cold-acclimation, but only moderately so in the roots of L.perenne.

Heterologously Expressed DaIRIPe7 Possesses RI Activity

Extract from E. coli expressing DaIRIPe was assayed for RI activity.Whilst extracts from cells expressing a putative D. antarcticaorthologue of histone H3.2 at a concentration of 400 μg mL⁻¹ of proteinpossess no RI activity, those expressing DaIRIPe7 retain activity downto 100 μg mL⁻¹ (FIG. 7). Therefore activity to inhibit further growth ofice crystals following freezing is specifically conferred by DaIRIPe7and can account, in some part, for this activity in planta.

EXAMPLE 3

Discussion

A physiological and functional genomics study in D. antarctica hasresulted in the identification and characterisation of a gene familyencoding IRIPs, the actions of which can account for its tolerance offreezing. D. antarctica has activity induced by cold acclimation, andpresent in the apoplasm, to inhibit ice recrystallisation therebyminimising the catastrophic plasmolytic consequences of uncontrolled icecrystal growth. This capacity is correlated with the expression of IRIPgenes, and the primary structure, conformation, localisation, and mostsignificantly, the activity of their products.

IRIP Genes Encode Proteins with Two Types of Potential Ice BindingDomains

We have isolated and characterised putative full length IRIP genes fromboth D. antarctica and L. perenne. The form reported previously from L.perenne (Sidebottom et al (2000) Nature 406:256), isolated as a proteinassociated with RI activity, lacked an N-terminal methionine, and wascomprised solely of 16 IRIP repeats. The IRIP forms reported here,unlike any other known AFPs, include 2 ice binding domains, the IRIP andLRR domains. Ten LRRs are also the predominant feature of an unrelated(FIG. 2D) ice recrystallisation inhibiting AFP from D. carota (Worrallet al. (1998) Science 282:115; Meyer et al. (1999) FEBS Lett. 447:171).

Although the LRR domain has the potential to function in ice binding, inthe various IRIP forms described here, there is a wide range ofvariation in its relative contribution to the overall primary structure.Thus whilst all IRIPs contain 16 IRIP repeats, LRRs are present from asmany as 9 iterations in the H. vulgare form HvIRIP to as few as one inthe D. antarctica forms, and in the extreme case of DaIRIPd the residueswith similarity to the LRR number only 17 of the usual complement of 24or 25 residues (FIG. 2A, B, C).

Despite the apparent plasticity in the number of LRRs, and even theirdispensability, other features commonly found in LRR proteins, includingthe region predicted to participate in 2 disulphide bridges, and theprobable signal sequence are invariant in IRIPs, suggesting that theyare important for structure/function and/or localisation.

What is Evolutionary Origin of IRIP Genes?

All plant (and animal) AFPs characterised to date appear to have arisenrelatively recently in evolutionary terms by the co-option of existingprotein structures (Logson and Doolittle (1997) Proc Natl Acad Sci USA.94:3485). This is also true of the IRIPs characterised in this study.Outside the IRIP domain itself IRIPs are structurally related toLRR-RLKs, having greatest affinity with orthologues of PSKR, a receptorfor the plant hormone phytosulfokine. PSKR first isolated andcharacterised in Daucus carota (carrot) consists of an extracellulardomain containing 21 LRRs, a single pass transmembrane domain, and acytoplasmic serine-threonine kinase domain (Matsubayashi et al. (2002)Science. 296:1470). Phytosulfokine is a secreted 5 residue sulfatedpeptide with a key role in cellular de-differentiation andredifferentiation (Matsubayashi and Sakagami (1996) Proc Natl Acad SciUSA. 93:7623). It is difficult to conceive the mechanistic connectionbetween hormonal regulation of cell fate determination and antifreezeactivity. A more likely scenario is that a PSKR related protein wasco-opted as an AFP, either because of intrinsic structuralcomplementarity to ice crystals, but more credibly as a vehicle totarget the “hitch hiking” IRIP domain to the cellular compartment whereRI activity is critical, the apoplast. It is possible to envisage anevolutionary scenario whereby a PSKR-like LRR-RLK protein has acquired anovel domain, the IRIP repeat domain, in the process losing itstransmembrane and intracellular kinase domains, thereby becoming anuntethered apoplastic protein with a novel function. Moreover PSKR-likegenes might have been predisposed for such a role because of theirexpression in organ primordia, cells of which are particularlyvulnerable to freezing induced damage. Like the IRIPs, the majority ofknown plant AFPs are derived from secreted proteins, many beingorthologues of pathogenesis-related proteins (Griffith and Yaish (2004)Trends Plant Sci. 9:399), and one, the AFP from D. carota (Worrall etal. (1998) Science 282:115; Meyer et al. (1999) FEBS Lett. 447:171) alsobeing related to LRR-RLKs.

IRIPs exhibit plasticity in the number and arrangement of LRRs, more sowhen compared to their presumed nearest relatives the PSKRs. Thus, thereare 17 LRRs in D. carota PSKR and its presumed homologue in O. sativa, 9in HvIRIP, 3 in the L. perenne and T. aestivum IRIP versions, and one,or part of one, in the D. antarctica forms (FIG. 2B, C). Suchevolutionary plasticity in number and arrangement of LRRs has been notedin analyses of LRR-RLKs (see for example Dixon et al. (1998) Plant Cell.10:1915)). All the LRRs in extant IRIP forms have high levels ofidentity and conservation with LRRs in the PSKRs, with no evidence forthe addition of any other sequences, LRR or otherwise. Therefore inrespect of the principle of maximum parsimony it is most likely that theevolution of IRIPs has resulted from the progressive loss of LRRs ratherthan their acquisition or rearrangement.

Because of their evolutionarily recent co-option as AFPs all known plantAFPs exhibit in their sequences clear affinities to particular classesof protein. Although this is true for the PSKR affinities of the LRRrelated N-terminal part of the IRIPs, the other potential ice bindingmotif in IRIPs, the IRIP repeat, exhibits no sequence similarity to anyreported nucleotide or amino acid sequences. For this reason its originis unknown. Because of its length, the shortest repeat known in an AFP,the IRIP repeat could conceivably be derived from a simple repetitiveelement of as few as 21 nucleotides in length, such as those found inintergenic DNA. However to date BLASTN searches have failed to identifyany closely related genomic sequences.

Genes encoding IRIP orthologues appear to be monophyletic in origin andconfined to the sub-family Pooideae. No sequences related to the IRIPdomain have been found in sequence similarity searches of any dicotsincluding Arabidopis thaliana, in the genome of O. sativa, or theextensive EST resources derived from Zea mays or Sorghum bicolor.Furthermore the IRIP clade (FIG. 3B) is highly distinct and deeplyrooted supporting the notion that IRIPs arose once early in theevolutionary history of the Pooideae and have subsequently diverged inboth copy number and structure. On this basis IRIP genes are predictedto have arisen sometime after the divergence of the Pooideae andPanicoideae 60 mya, but before that of the Triticodae and Poodae 35 mya(Huang et al, (2002) Plant Mol Biol. 48:805).

Structural Modelling Predicts that IRIP Repeat has Greater Affinity forIce than LRR

We have used comparative homology modelling to devise a theoretical 3-Dstructure for full length IRIPs. A truncated version of LpIRIP modelledpreviously (Kuiper et al (2001) Biophys. J. 81: 3560) did not includethe LRR domain, nor the twin disulphide bond-forming N-terminal domain.The structural model demonstrates that both the IRIP and LRR domains cancontribute to a common, structurally complementary ice-binding domain.

The predominant ice-binding region is predicted to be the IRIP domain,which presents two ice-binding faces, on either side of the β-rolldomain. The putative ice binding surfaces however are not as regular asthe stereotypical threonine-X-threonine motifs in beta-rollconfigurations observed in two unrelated insect AFPs with high THactivity (Graether at al (2000) Nature. 406:325; Liou at al, (2000)Nature. 406:322). The D. antarctica and L. perenne IRIPs exhibit only 30to 40% threonine at the analogous positions. This is likely due to thedifferences in the primary function of the proteins. Insect AFPs mustprovide appreciable TH activity as most insects are not freeze-tolerant.The regularity of the threonine residues on the presenting ice bindingsurfaces has been implicated in their high TH activity, TH activityhaving been shown to rapidly decrease with increasing mutationalsubstitution of residues in the ice binding surface (Marshall et al(2002) FEBS Lett. 529:261). By contrast since D. antarctica is freezetolerant the primary purpose of AFPs in this organism would be toprovide RI activity, to avoid the plasmolytic consequences of continuedice crystal growth in already frozen tissue.

In fact, IRIPs may have evolved to have low TH activity as high activitymay prove detrimental during the inevitable seasonal freezing of theseplants. If a plant were to deploy an IRIP with a relatively high THactivity, the apoplastic fluid of the plant would remain liquid untilthe temperature dropped below the lower end of the TH gap. Freezingwould then occur much more rapidly than if initiated close to thefreezing equilibrium point, and would do so with the spicular dendriticgrowth observed with other AFPs, potentially doing much mechanicaldamage to cells.

The LRR domains of full-length IRIPs are also predicted to contribute toice binding surfaces but not with the inherent structural complementaryto the prism face of ice of the IRIP domain. Whilst solved crystalstructures of LRRs in proteins form parallel β-sheets on one side of aβ-roll, the other side is made up of adjacently packed α-helical strands(Di Matteo et al (2003) Proc. Natl. Acad Sci. USA 100: 10124). As theα-helical regions cannot pack as closely as the β-sheet regions the βroll structure will curve, displaying a concave β-sheet region,proportional to the numbers of LRRs. Extended curved β-sheet surfaces ofLRR regions do not present an optimal surface lattice match to ice,although the AFP from D. carota consists predominantly of 10 LRRs.Indeed globular type Ill fish AFP also does not have an obvious regularice binding surface and yet displays reasonable TH and RI activity(Baardsnes and Davies, (2002) Biochim Biophys Acta. 1601:49).

Genetic Mapping of LpIRIP and Relationship to Syntenic Cold Toleranceand Vernalisation QTLs

In order to determine the location of IRIP genes in the genome of L.perenne and their proximity to endogenous or syntenic cold tolerance andvernalisation quantitative trait loci (QTLs) LpIRIPb was geneticallymapped using single nucleotide polymorphism (SNP).

The analysis of SNP variation in LpIRIP gene(s) revealed a high level ofvariation, even compared to average values observed over a large sampleof perennial ryegrass. This observation, along with the excess ofrecovered haplotype structures, is strongly suggestive of multiple genestructure. Although Southern hybridisation analysis indicated arelatively simple genome organisation (FIG. 5C), a number of minor bandswere observed, which may correspond to paralogues of the LpIRIPa gene.The segregating LpIRIPNA476 SNP locus may identify such a paralogoussequence, based on the results of comparative genetics and genomicsanalysis. Macrosynteny based on heterologous RFLP markers hasdemonstrated a broad correspondence between each of the perennialryegrass linkage groups and each of the homoeologous groups of wheat(Jones et al. (2002) Theoretical and Applied Genetics 105: 577). On thisbasis, the SNP locus location on perennial ryegrass LG1 shouldcorrespond to a region of conserved synteny with the wheat homoeologous1S chromosomes. However, comparative genomics has identified wheatsequences assigned to the group 4L and 5L chromosomes, which are relatedthrough evolutionary translocations (Devos et al., (1995) Theoreticaland Applied Genetics 91:282). The location of LpIRIP ortholoci onTriticeae group 5 chromosomes is also consistent with the detection ofQTLs for winter hardiness and frost tolerance on these chromosomes inwheat (Sutka (1994) Euphytica 77:277; Galiba et al., (1995). Theoreticaland Applied Genetics 90:1174; Galiba et al., (1997) Theoretical andApplied Genetics 95:265; Toth et al. (2003) Theor Appl Genet. 107:509)and barley (Pan et al. (1994) Theoretical and Applied Genetics 89: 900;Francia et al. (2004) Theoretical and Applied Genetics 108: 670;Reinheimer et al, (2004) Theoretical and Applied Genetics 109: 1267), inthe same region as the vernalisation response genes that control headingdate. Based on conserved synteny, this would predict a location inperennial ryegrass on the upper part of LG4 (Yamada et al. (2004) CropScience 44: 925). The detection of wheat IRIP paralogues on chromosome1BL, however, indicates the complexity of this gene family in wheat, andsuggests that paralogous sequences may be located on other LGs inperennial ryegrass as well. In this interpretation, the non-segregatingSNPs may identify variation between LG1 and LG4-located paralogues. Inorder to test this hypothesis, it would be necessary to identifypolymorphic SNPs for the second gene copy in other germplasm. Anotherpossibility is that the LG1-located xLpiripna476 locus identifies anon-syntenic region. The ends of each LG in perennial ryegrass wereenriched for non-syntenic markers, as previously shown for other Poaceaespecies (Jones et al (2002) Theoretical and Applied Genetics 105:577).The closest marker to xLpiripna476 is an EST-RFLP marker, xLpesi3f(Faville et al., Theor Appl Genet. in press), which preferentiallydetected wheat ESTs allocated to deletion bins on chromosome 4A.Finally, it is to be understood that various alterations, modificationsand/or additions may be made without departing from the spirit of thepresent invention as outlined herein.

Documents cited in this specification are for reference purposes onlyand their inclusion is not acknowledgment that they form part of thecommon general knowledge in the relevant art.

1. The isolated nucleic acid or nucleic acid fragment of claim 27,wherein the plant is a Deschampsia species.
 2. The isolated nucleic acidor nucleic acid fragment according to claim 1 wherein said Deschampsiaspecies is Deschampsia antarctica.
 3. (canceled)
 4. (canceled) 5.(canceled)
 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. Anucleic acid construct including one or more nucleic acids or nucleicacid fragments according to claim
 27. 11. (canceled)
 12. (canceled) 13.(canceled)
 14. (canceled)
 15. A plant cell, plant, plant seed or otherplant part, including a construct according to claim
 10. 16. (canceled)17. A method of modifying tolerance of freezing and/or low temperaturestress in a plant, the method including introducing into the plant aneffective amount of a nucleic acid or nucleic acid fragment according toclaim
 27. 18. (canceled)
 19. A substantially purified or isolatednucleic acid or nucleic acid fragment including a single nucleotidepolymorphism (SNP) from a nucleic acid fragment according to claim 27.20. (canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled) 24.(canceled)
 25. (canceled)
 26. (canceled)
 27. An isolated nucleic acid ornucleic acid fragment encoding an ice recrystallization inhibitionprotein derived from a plant, wherein the protein includes: an IRIPdomain including 3 to 32 repeats of the amino acid sequence set forth inSEQ ID NO: 141; and an amino acid residue motif as set forth in SEQ IDNO:
 145. 28. The isolated nucleic acid or nucleic acid fragment of claim27 wherein the nucleic acid comprises a nucleotide sequence set forth inany of SEQ ID NOs: 1-16, 18-21, 23-30, 32-37, 39-45, or 47-53.
 29. Anucleic acid construct including one or more nucleic acids according toclaim
 27. 30. The isolated nucleic acid of claim 27, wherein the proteinincludes an amino acid sequence selected from the list consisting of:SEQ ID NOs: 17, 22, 31, 38, 46 and
 54. 31. The isolated nucleic acid ofclaim 27 wherein the IRIP domain includes 15 or 16 tandem repeats of theamino acid sequence set forth in SEQ ID NO:
 141. 32. The isolatednucleic acid of claim 27 wherein the protein further includes three orfewer Leucine Rich Repeat (LRR) motifs.
 33. The isolated nucleic acid ofclaim 32 wherein the LRR motif(s) are positioned N-terminal to the IRIPdomain.
 34. The isolated nucleic acid of claim 32 wherein the amino acidresidue motif as set forth in SEQ ID NO: 145 is positioned N-terminal tothe LRR motif(s).
 35. The isolated nucleic acid of claim 27 wherein theprotein includes one or fewer LRR motifs.
 36. The isolated nucleic acidof claim 27 wherein an LRR motif includes the amino acid sequence setforth in SEQ ID NO: 128 or SEQ ID NO:
 143. 37. An isolated proteinencoded by the nucleic acid of claim
 27. 38. A method of modifyingtolerance of freezing and/or low temperature stress in a plant, themethod including introducing into the plant an effective amount of aconstruct according claim 10.